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Pasteurella multocida (P. multocida) is an important zoonotic pathogen. In addition to lung lesions, necropsies have revealed macroscopic lesions in the heart in clinical cases. However, most previous studies focused on lung lesions while ignoring heart lesions. Therefore, to investigate the immune response of the P. multocida-infected heart, two murine infection models were established by using P. multocida serotype A (Pm HN02) and D (Pm HN01) strains. Histopathological examination revealed heterogeneous inflammatory responses, including immune cell infiltration in the epicardial and myocardial areas of the heart. Transcriptome sequencing was performed on infected cardiac tissues. To explore the traits of immune responses, we performed the functional enrichment analysis of differentially expressed genes, gene set enrichment analysis and gene set variation analysis. The results showed that the innate immune pathways were significantly regulated in both groups, including the NOD-like receptor signaling pathway, the complement and coagulation cascade and cytokine–cytokine receptor interaction. The Toll-like receptor signaling pathway was only significantly activated in the Pm HN02 group. For the Pm HN02 group, immunohistochemistry analysis further verified the significant upregulation of the hub component MyD88 at the protein level. In conclusion, this study reveals critical pathways for host heart recognition and defense against P. multocida serotypes A and D. Moreover, MyD88 was upregulated by P. multocida serotype A in the heart, providing a theoretical basis for future prevention, diagnosis and treatment research. [ABSTRACT FROM AUTHOR]

Pasteurella multocida (P. multocida) is an opportunistic pathogen that is common in livestock and poultry and leads to massive economic losses in the animal husbandry sector. In this study, we challenged mice with P. multocida strain HN02 by intraperitoneal injection and collected spleens to measure bacterial loads. We also performed histopathological analysis by hematoxylin and eosin (H&E) staining. Then we used RNA-sequencing (RNA-seq) to detect the mRNA expression levels in the mouse spleen and quantitative real-time PCR (qRT-PCR) to verify the sequencing data. Finally, we examined the effect of HN02 on anti-inflammatory cytokine interleukin-10 (IL-10) protein expression in the spleen through immunohistochemical analysis. The results showed that compared to those in the control group, the mouse spleens in the challenge group had lesions, and the average bacteria loads was (3.07 ± 1.09) × 106 CFU (colony-forming unit)/g. The RNA-seq results determined 3653 differentially expressed genes (DEGs), and the qRT-PCR analysis revealed immune-related genes consistent with the expression trend in the sequencing data. The number and area of IL-10 positive cells substantially increased to resist inflammation in the challenge group. In conclusion, we analyzed the spleens of mice infected with P. multocida from multiple perspectives, and our findings lay a foundation for subsequent studies on the mechanism of pathogen-host interactions. [ABSTRACT FROM AUTHOR]

Simple Summary: Pasteurella multocida (Pm) infection can cause pasteurellosis in goats. Currently, the mechanisms of Pm infection in goats are limited. In this study, the effects of Pm infection to goats were characterized from the aspects of histopathology, miRNA and mRNA expression dynamics in lung of goats infected for 1, 2, 5 and 7 days and the clinical symptoms of animals. We further revealed the potential role of differentially expressed RNAs, which are commonly involved in and regulate the host immune reaction to the Pm infection. The differentially expressed genes were mapped to marker genes of the immune cells in the lung, which further confirmed the cells detected by the histopathological method. Pasteurella multocida (Pm) infection causes severe respiratory disease in goats. We investigated the effects of the Pm infection intratracheally on the histopathology, miRNA and mRNA expression dynamics in the lung of goats infected for 1, 2, 5 and 7 days. Pm infection caused fever, which significantly (p < 0.05) increased the body temperature of the goats from day 1 to 5. Haemotoxylin–eosin staining of the infected lung tissue showed characteristics of suppurative pneumonia with inflammatory cells infiltration and the lung structure destruction. During the Pm infection of the goats, compared with the control group, there were 3080, 3508, 2716 and 2675 differentially expressed genes and 42, 69, 91 and 108 significantly expressed miRNAs (|log2Fold Change| > 1, p < 0.05) in the Pm_d1, Pm_d2, Pm_d5 and Pm_d7 groups, respectively. Five miRNAs and nine immune-related genes were selected for confirmation by reverse transcription–polymerase chain reaction. The results indicated that the expression patterns of the miRNAs and genes were consistent with those determined by next-generation sequencing. The differentially expressed genes were enriched in cytokine–cytokine receptor interaction, cell adhesion molecules, complement and coagulation cascades, tight junction and phagosome Kyoto Encyclopedia of Genes and Genomes pathways and cytokine production, leukocyte migration, myeloid leukocyte migration, cell periphery, plasma membrane, extracellular region part, extracellular region and other Gene Ontology terms. The differentially expressed genes were mapped to marker genes in human and mouse lung cells. The results showed the presence of some marker genes of the immune cells. Compared with the CK group, five miRNAs and 892 common genes were differentially expressed in the Pm_d1, Pm_d2, Pm_d5 and Pm_d7 groups. The target relationships between the common 5 miRNAs and 892 differentially expressed genes were explored and the miRNAs involved in the host immune reaction may act through the target genes. Our study characterized goats' reaction in the lung from histopathological and molecular changes upon Pm infection, which will provide valuable information for understanding the responses in goats during Pm infection. [ABSTRACT FROM AUTHOR]

Brucella-caused brucellosis is one of the most widespread worldwide zoonoses. Lipopolysaccharide (LPS) of Brucella, which functions as pathogen-associated molecular patterns (PAMPs), is an important virulence factor that elicits protective antibodies. Per of B. melitensis is involved in the biosynthesis of the O-side chain of LPS. Autophagy is a crucial element of the innate immune response against intracellular pathogens including Brucella. In this study, we observed that autophagy was inhibited in RAW264.7 cells infected with Brucella melitensis ∆per. And, a high-throughput array-based screen and qRT-PCR validation were performed to identify the differentially expressed miRNAs in RAW264.7 cells infected with B. melitensis M5-90 ∆per. The results suggested that mmu-miR-146a-5p, mmu-miR-155-5p, mmu-miR-146b-5p, and mmu-miR-3473a were upregulated and mmu-miR-30c-5p was downregulated. During B. melitensis M5-90 ∆per infection, the increased expression of miR-146b-5p inhibited the autophagy activation in RAW264.7 cells. Using a bioinformatics approach, Tbc1d14 was predicted to be a potential target of miR-146b-5p. The results of a luciferase reporter assay indicated that miR-146b-5p directly targeted the 3′-UTR of Tbc1d14, and the interaction between miR-146b-5p and the 3′-UTR of Tbc1d14 was sequence-specific. High-throughput RNA-Seq-based screening was performed to identify differentially expressed genes in Tbc1d14-expressing RAW264.7 cells, and these were validated by qRT-PCR. Among the differentially expressed genes, four autophagy associated genes, IFNγ-inducible p47 GTPase 1 (IIGP1), nuclear receptor binding protein 2 (Nrbp2), transformation related protein 53 inducible nuclear protein 1 (Trp53inp1), and immunity-related GTPase family M member 1 (Irgm1), were obtained. Our findings provide important insights into the functional mechanism of LPS of B. melitensis. [ABSTRACT FROM AUTHOR]

Melioidosis is a severe and fatal tropical zoonosis, which is triggered by Burkholderia pseudomallei. To better understand the host’s response to infection of B. pseudomallei, an RNA-Seq technology was used to confirm differentially expressed genes (DEGs) in RAW264.7 cells infected with B. pseudomallei. In total, 4668 DEGs were identified across three time points (4, 8, and 11 hours after infection). Short Time-Series Expression Miner (STEM) analysis revealed the temporal gene expression profiles and identified seven significant patterns in a total of 26 profiles. Kyoto Encyclopedia of Genes and Genomes (KEGG) was utilized to confirm significantly enriched immune process-associated pathways, and 10 DEGs, including Ccl9, Ifnb1, Tnfα, Ptgs2, Tnfaip3, Zbp1, Ccl5, Ifi202b, Nfkbia, and Nfkbie, were mapped to eight immune process-associated pathways. Subsequent quantitative real-time PCR assays confirmed that the 10 DEGs were all upregulated during infection. Overall, the results showed that B. pseudomallei infection can initiate a time-series upregulation of immune process-associated DEGs in RAW264.7 macrophage cells. The discovery of this article helps us better understand the biological function of the immune process-associated genes during B. pseudomallei infection and may aid in the development of prophylaxis and treatment protocols for melioidosis. [ABSTRACT FROM AUTHOR]

Pasteurella multocida (P. multocida) is a highly infectious, zoonotic pathogen. Outer membrane protein A (OmpA) is an important virulence component of the outer membrane of P. multocida. OmpA mediates bacterial biofilm formation, eukaryotic cell infection, and immunomodulation. It is unclear how OmpA affects the host immune response. We estimated the role of OmpA in the pathogenesis of P. multocida by investigating the effect of OmpA on the immune cell transcriptome. Changes in the transcriptome of rat alveolar macrophages (NR8383) upon overexpression of P. multocida OmpA were demonstrated. A model cell line for stable transcription of OmpA was constructed by infecting NR8383 cells with OmpA-expressing lentivirus. RNA was extracted from cells and sequenced on an Illumina HiSeq platform. Key gene analysis of genes in the RNA-seq dataset were performed using various bioinformatics methods, such as gene ontology enrichment analysis, Kyoto Encyclopedia of Genes and Genomes enrichment analysis, Gene Set Enrichment Analysis, and Protein–Protein Interaction Analysis. Our findings revealed 1340 differentially expressed genes. Immune-related pathways that were significantly altered in rat alveolar macrophages under the effect of OmpA included focal adhesion, extracellular matrix and vascular endothelial growth factor signaling pathways, antigen processing and presentation, nucleotide oligomerization domain-like receptor and Toll-like receptor signaling pathways, and cytokine-cytokine receptor interaction. The key genes screened were Vegfa , Igf2r , Fabp5 , P2rx1 , C5ar1 , Nedd4l , Gas6 , Cxcl1 , Pf4 , Pdgfb , Thbs1 , Col7a1 , Vwf , Ccl9 , and Arg1. Data of associated pathways and altered gene expression indicated that OmpA might cause the conversion of rat alveolar macrophages to M2-like. The related pathways and key genes can serve as a reference for OmpA of P. multitocida and host interaction mechanism studies. • P. multocida OmpA was transcriptionally overexpressed in rat alveolar macrophages. • Dysfunction and polarization of alveolar macrophages. • Significant functional changes in tissue repair, angiogenesis and immunosuppression were found in RNA-seq bioinformatics analysis. [ABSTRACT FROM AUTHOR]

Abstract: Brucellosis, caused by Brucella spp., is an important disease affecting not only human health, but also a number of animal species around the world. A receptor for LPS of Brucella and important innate immune molecule, CD14, has been implicated in the initiation of the inflammatory response to sepsis. Evidence indicates that upstream inhibition of the LPS initiated inflammatory pathway is an effective therapeutic approach for attenuating damaging immune activation. The aim of this study was to explore the possibility of using RNA interference (RNAi) targeting mCD14 as a strategy for inhibiting the secretion of tumor necrosis factor alpha (TNFα) and the production of nitric oxide (NO) from Brucella-stimulated RAW264.7 cells and attenuating damaging immune activation. The current study stably incorporated mCD14-shRNA-224 into the RAW264.7 cell line by lentiviral gene transfer to successfully knockdown mCD14, and was then challenged with Brucella melitensis M5-90. The secretion of TNFα, interleukin (IL)-12, CXCL1/KC, and inducible nitric oxide synthase (iNOS) protein expression, and NO production were evaluated. The mCD14-shRNA-224 knockdown was shown to effectively inhibit B. melitensis M5-90-stimulated TNFα release, iNOS protein expression, and NO production, but no significant differences were observed for IL-12 and CXCL1/KC. These findings provide the basis for the development of RNAi-based prophylaxis and therapy for B. melitensis induced inflammatory disease. [Copyright &y& Elsevier]